Study on the differences in aroma components and formation mechanisms of “Nasmi” melon from different production areas

Abstract Aroma is an important factor that guides consumers in purchasing and is thus very important in melon research. To our knowledge, the number of studies with a focus on the aroma differences of the same melon variety in different production areas is largely limited. In this study, the differences in aroma components of “Nasmi” melons from two different production regions were analyzed using gas‐phase ion migration spectroscopy. Transcriptome sequencing was performed for analyzing fragrance‐related genes. Results showed that there were significant differences in the aroma components between products from the two regions. The total amount of aroma compounds from the Turpan region (TT) was 1.7 times higher than that from the Altay region (AT). Through the analysis of transcriptome data, the key genes encoding melon aroma components in different regions were identified as ethanol dehydrogenase, 3‐hydroxyl‐coenzyme A (CoA) dehydrogenase, acyl‐CoA oxidase, long‐chain acyl‐CoA synthetase, acetaldehyde dehydrogenase, and acetyl‐CoA acyltransferase. Real‐time quantitative polymerase chain reaction (RT‐qPCR) showed that the verified genes were similar to the transcriptome. In this study, the main aroma components of the same variety of melon that differed in different production areas and the key genes causing these differences were identified. In addition, the aroma metabolic pathway of melon in different regions was preliminarily elucidated. These results could provide a theoretical basis for further study of the formation mechanism of melon aroma and breeding.


| INTRODUC TI ON
Melon (Cucumis melo L.) is a gourd family (Cucurbitaceae) cucumber (Cucumic) annual trailing herb and is grown on five continents worldwide. According to the FAOSTAT (Food and Agriculture Organization Statistical Database) published by the Food and Agriculture Organization (FAO) of the United Nations, the world's total melon production in 2019 was 27.5013 million tons. China's melon production was 13.5414 million tons, accounting for 49.24% of the world's total production. Xinjiang is one of the main production areas in China, with the melon production ranking first in China year-round (Xiong et al., 2018). Melon fruit is favored by consumers because of its juicy pulp and unique aroma. Flavor (aroma and taste), color, texture, and nutrients are the main quality determining factors of melon, among which the flavor and color play a leading role in melon consumption (Obando-Ulloa, Jowkar, et al., 2009). Therefore, study on fruit aroma is very important in melon breeding and quality control. It has been receiving increasing attention from breeders and researchers. The main aroma compounds of melon and their metabolic pathways have been reported by other research groups (Shi et al., 2020).
It has been reported that the aroma of melon fruit is related to development period, variety, postharvest storage, and production area. Beaulieu et al. determined the volatile components of cantaloupe at different developmental stages using headspace solid-phase microextraction--gas chromatography-mass spectrometer (HS-SPME-GC-MS) technology (Beaulieu & Grimm, 2001). They found that the aroma of cantaloupe varied greatly during different developmental stages, and most ester compounds gradually increased with increasing maturity. Lamikanra et al. found that the most prominent volatile compounds in melon were methyl-butyl acetate and hexyl acetate when stored at 4°C. These two compounds, which contribute to the fruit aroma characteristics of many fruits, are usually present in relatively large proportions in Hami melon (Lamikanra & Richard, 2002).
Elazar Falik et al. measured the aroma of two different varieties of melon in Israel and found that C8 fruit had higher levels of aroma volatiles than 5080 fruit (Fallik et al., 2001). Xiao Z et al. determined and analyzed the volatile components of the melon varieties such as Jiashigua, Xizhou Mi 17, and Minqin from different production regions.
The main identified metabolic pathways of melon aroma include the fatty acid pathway, amino acid pathway, secondary metabolic pathway, and conversion of alcohols and aldehydes into esters (Lewinsohn et al., 2001;Schwab et al., 2008;Tang et al., 2015). In the fatty acid pathway, straight-chain aliphatic alcohols, aldehydes, ketones, and esters can be synthesized. Saturated fatty acids are catalyzed by β-oxidation and acyl-CoA oxidase to produce lactones.
In addition, some unsaturated fatty acids are directly oxidized to form C6 aldehydes and corresponding alcohols and esters by lipoxygenase (LOX). In the amino acid pathway, branched-chain alcohols and esters are formed mainly through the action of transaminase and dehydrogenase. Aldehydes produced in the above two pathways generate alcohols under the action of alcohol dehydrogenase (ADH). In combination with acyl-CoA, corresponding esters can be formed under the action of alcohol acyltransferase (AAT). In secondary metabolic pathways, synthesis of some melon volatile phenols and terpenoid substances via the shikimic acid pathway is one of the most important branches.
With the combination of these various aroma metabolism pathways, melon forms a unique and strong aroma that appealing consumers love. Related studies have found that enzymes related to melon aroma metabolism mainly include lipoxygenase (LOX), alcohol dehydrogenase (ADH), alcohol acyltransferase (AAT) acyl-coenzyme A oxidase, aldehyde dehydrogenase, and others (Buchhaupt et al., 2012;Gur et al., 2016;Li et al., 2016a;Shalit et al., 2001).
With rich aroma compound content, "Nasmi" melon which was selected and bred by Academician of Mingzhu Wu was used as the model fruit in our study. Gas chromatography-ion mobility spectrometry (GC-IMS) technology was used to determine and analyze the aroma components of melons in different production areas.
RNA-sequencing (RNA-seq) technology was used to carry out highthroughput sequencing on fruits of the same "Nasmi" melon from different production areas in Xinjiang. Based on the sequencing data, the genes related to melon fruit aroma metabolism were identified.
The formation mechanisms of melon aroma were also subsequently analyzed. These studies may contribute to more fundamental research in melon aroma and industrial practices.

| Materials
The sampling area is shown in Table 1. The melons with same planting patterns from Turpan are provided by the Xinjiang and Altay experimental base. The same batches of seeds were provided by the Xinjiang Academy of Agricultural Sciences Research Center. Melons were sampled 43 days after pollination. Thirty melons with moderate maturity and no disease/insect pests were selected in the two test regions respectively. Samples were stored at −80°C until use.
Three biological replicates were obtained for each sample. The test sample "Nasmi" was planted in two experimental bases, with similar treating and environmental conditions, such as seed pretreatment, plant spacing, seedling stage management, fertilizer, and watering.

| Melon aroma composition determination
Volatile components were determined by the GC-IMS technology (Wang, Chen, & Sun, 2019). An automatic headspace (HS) device was used to extract samples. Three-gram sample was placed in a 20 ml headempty bottle with a magnetic cap, followed by an incubation at 50°C for 15 min. The rotating speed was set to 500 rpm (revolutions per minute). Three hundred microliter sample was injected each time with the injection needle temperature of 55°C. The GC was equipped with a FS-SE-54-CB-1 capillary column (15 m × 0.53 mm ID, 1 μm). The column temperature stayed at 60°C during the process.
The running time was set to 30 min. Nitrogen (99.99%) was used as a carrier gas and its flow rate was initially set at 2 ml/min for 2 min, then increased to 100 ml /min within 18 min and held for 10 min. The 9.8 cm drift tube was operated at constant temperature (45°C) and voltage (5 kV). The flow rate of the drift gas (nitrogen) was set to 150 ml/min. Volatile compounds were identified by comparing the retention index (RI) and the drift time (DT) (the time it takes for ions to reach the collector through drift tube, in milliseconds) of standard in the GC-IMS library.

| Transcriptome sequencing
Total RNA extraction from melon fruit Total RNA extraction was carried out according to the kit operation manual. After extraction, the RNA integrity was detected by an Agilent 2100 biological analyzer.

Library preparation and sequencing
Following magnetic bead enrichment of messenger RNA mRNA with Oligo(dT) poly (A), RNA was obtained in segmentation buffer using random N6 primers for reverse transcription, according to the instructions for the retrovirus kit. After that, second strand complementary DNA (cDNA) was synthesized to form double-stranded DNA. The double-stranded DNA underwent phosphorylation at the 5′ end and 3′ end to form the sticky end of an "A". The 3′ end had a bulge in the "T" drum bubble joint, which facilitated connection of the product through PCR amplification with specific primers into a single PCR product following thermal denaturation. Then bridgetype primers facilitated cyclization of single-stranded circular DNA to create a single-strand DNA library. Finally, the DNBSEQ platform was used for sequencing.

| qRT-PCR verification
DNA extraction and reverse transcription were performed according to the instructions of kit. The reaction system was prepared using cDNA as template according to the SuperReal PreMix Plus (SYBR Green) kit. As shown in Table 2, the system volume was 10 μl.
Each gene was prepared and analyzed in triplicate. The target gene and internal reference gene were placed in the same plate and measured simultaneously. According to the PCR amplification reaction and melting temperature (Tm) values provided by internal reference genes and target genes, the real-time fluorescence PCR conditions were set as follows: predenaturation at 94°C for 5 min followed by a cycle with 10S denaturation at 94°C, 30S annealing at 72°C, 15 S extensions, 40 cycles, and 57-95°C melting curve plotted with a heating rate of 1°C/min. The obtained detection results were processed by the 2 −ΔΔCT method (Livak & Schmittgen, 2001). The relative contents of multiple differential genes in the same melon fruit from different production areas were calculated to analyze their differential expression. Primer information for 11 different genes designed using Primer 5.0 software is shown in Table 3.

| Transcriptome data analysis
Sequence quality control and cleaning SOAPnuke V1.5.2 software (https://github.com/BGI-flexl ab/ SOAPnuke) was used for filtering and statistical processing of the raw data. The steps were as follows: (I) Removal of reads containing joints (joint contamination); (II) removal of reads with unknown base N content greater than 5%; (III) removal of low-quality reads. Clean reads were obtained and stored in FASTQ format for subsequent analysis.

Quantitative analysis and screening of differential gene expression
Gene expression level, also known as expression abundance, is the first and most important factor in transcriptome data analysis (Sonali et al., 2020). FPKM (fragments per kilobase of transcript per million fragments mapped) was used as an indicator to measure the expression levels of transcripts or genes. The calculation formula is as follows: In Otherwise, it was considered to be downregulated. The conditions for screening differential genes were that the differential multiple was more than 2 and the corrected p-value was less than or equal to .05 (Love et al., 2014).

Functional classification and annotation of differential genes
Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (GO and KEGG) annotation databases are by far the most widely used public databases. The phyper function in R software was used for enrichment analysis of differential genes. The GO classification annotation of differential genes enables us to understand the classification of biological functions of differential genes. The GO database (http://www.geneo ntolo gy.org/) notes mainly include biological processes, components of cells, and molecular functions. The KEGG database (https://www.kegg.jp/) system includes the functional systematic analysis of intracellular metabolic pathways and gene products, which is helpful for the study of complex gene biological behavior. Analyses on the obtained differential genes based on the KEGG database facilitate the understanding of major metabolic pathways which are demonstrating high gene expression. Figure 1 and Table 4 show the qualitative and quantitative results of volatile chemicals in melon samples from various production regions. Forty-seven signal peaks were detected by GC-IMS, from which 36 typical compounds were identified. The other 11 compounds got no qualitative results due to the limited data in the library database (Table 4). Based on the identified compounds, the volatile compounds in melon samples were esters (8), alcohols (7), aldehydes (10), ketones (2), pyrazines (1), terpenoids (1), and others. As shown in Figure 1 and shown in the purple box on the upper right of the map, the concentration of most ester substances in the AT sample was higher than that in the TT sample. Monomers and dimers are recognized in most ester products. According to the peak area, ethyl acetate was the most abundant ester compound in the samples from both production regions. There is no significant difference in ethyl acetate content between TT samples and AT samples. The contents of isobutyl acetate and ethyl propanoate in the AT sample were significantly higher than those in the TT sample. There are 7 alcohols, oct-1-en-3-ol, linalool, 3-methyl-butanol, (E)-2hexen-1-ol, 2-methyl-butanol, n-hexanol, and 2-propanol, which can be detected in this study. The contents of n-hexanol, (E)-2hexen-1-ol, and 2-propanol in the TT sample were significantly higher than those in the AT samples, while the content of linalool and 3-methylbutanol in the AT samples was significantly higher than that in the TT samples. Ten aldehyde compounds, (E)-2nonenal, (E, Z)-2,6-nonadienal, 5-methyl-2-thiophenecarboxaldeh yde, nonanal, octanal, heptanal, (E)-2-pentenal, 2-methylbutanal, 3-methylbutanal, and pentanal, were detected. As shown in the red box, the content of most aldehydes in the TT sample was higher than that in the AT sample. The 3-methylbutanal is the only aldehyde which showed a slightly higher content in AT samples than in TT samples. The content of (E)-2-nonenal in TT was 11.5 times higher than that in the AT sample. The contents of nonanal, octanal, (E, Z)-2, 6-nonadienal, and heptanal in the TT sample were significantly higher than those in the AT sample. Additionally, two different types of ketones, methyl-5hepten-2-one and 1-penten-3-one, were detected by our instruments. The content of methyl-5-hepten-2-one was higher in AT samples. The 1-penten-3-one content of TT samples was nearly seven times higher than that in AT samples. The contents of 2,5-dimethylpyrazine (pyrazine) and limonene (terpene) were higher in AT samples than in TT samples.

| Transcript mapping
Sequencing was performed on the TT samples and AT samples.
The obtained data are shown in Table 5. More than 1 million original reads were obtained from all the 6 samples. The filtered clean reads ratio was above 99%. The proportion of the number of bases with a quality value greater than 20 in filtered reads was above 97%. More than 91% of the bases have a mass value greater than 30.

| Differential gene identification
According to differential gene identification, a total of 9358 differentially expressed genes were found in "Nasmi" melon from the two regions, among which 4621 genes were upregulated and 4737 genes were downregulated. The obtained differentially expressed genes were used for subsequent analysis. As shown in Figure  As shown in Figure 2b, the obtained differentially expressed genes were classified into KEGG biological pathways. A total of 4061 differentially expressed genes were classified into different groups involved in 21 pathways. These groups of genes are mainly associated with certain biological functions, such as cellular processes, environmental information processing, genetic information processing, metabolism, organizational systems, and human disease. "Metabolism"  (1690), followed by the "carbohydrate metabolism" group (751) and "translate" group (628). Some other groups, such as "fold, classification and degradation," "transportation and catabolism," and "signal transduction" gene annotation, are also showing substantial differences.

| KEGG annotation pathway enrichment
As shown in Figure 3, Enrichment analysis showed that these dif-

| Genes related to aroma metabolism of melon from different production areas
Fatty acid metabolism in the melon aroma formation process plays a very important role. Transcriptional omics data showed that a total of five pathways related to the fatty acid metabolism pathway are involved. Pyruvate metabolism and a large amount of amino acid metabolism are also associated with aroma composition. The expression of genes enriched in some of the metabolic pathways related to aroma is shown in Table 6. Some representative differential genes are shown in Table 7.
Acetyl-CoA, the hub of various biological metabolism pathways, is also the precursor of the synthesis of aroma components. Its synthe- As shown in Figure 4, studies on aroma metabolic pathways have preliminarily demonstrated the metabolic pathway of melon fruit aroma (Beaulieu, 2006;Gonda et al., 2010;Lange et al., 2000;Mayobre et al., 2021;Song et al., 2021). The precursor substance pyruvate is synthesized from glycolysis or organic acid metabolism. Under the action of pyruvate decarboxylase (PDC), acetyl-CoA, which is crucial to aroma metabolism, is formed.

| Real-time quantitative RT-PCR verification
To verify the transcriptome data, we randomly selected 11 genes for real-time fluorescence quantitative verification. As shown in Table 8, annotated information, expression levels obtained from transcriptome data, and relative contents obtained using real-time fluorescence quantitative technology for 11 verified genes are presented. The results showed that the 11 verified genes were consistent with the transcriptome data, indicating the high reproducibility and reliability of transcriptome analysis. Under sufficient light conditions, the melon plant grows robustly.

| Influence of region on melon aroma components
There were significant differences in temperature, illumination, altitude, and other conditions between the products from two regions in this study. This is the main cause of the significant differences in aroma components of the same variety of "Nasmi" melon. Not all aroma ingredients are related to fragrance. Some characteristic aroma contents are very high, but it is difficult for us to smell be-

| Aroma metabolic main way
In recent years, research on aroma has attracted great attention In the fatty acid pathway, pyruvate is an important precursor  (Wang, Zhang, et al., 2019). Liu et al. (Liu et al., 2020) resequenced 297 melon materials to reveal the genome improvement history of melon and the loci related to fruit traits. This study found that the Cm AAT gene was a special gene in melon fruits and was closely related to aroma formation. CM AATS plays an important role in the last step of ester biosynthesis, leading to the synthesis of various ester aromas (Chen et al., 2016).
The high content of alcohols and volatile esters in melon is related to the activity of CMAAT (Melo3C024771), which determines the high content of alcohols and volatile esters in melon volatiles (Galaz et al., 2013). This study showed that the action of AAT produces alcohols and acetyl in combination with acetyl coenzyme A, resulting in esters. Additionally, this study found that alcohol and aldehyde compounds result in the transformation of esters. In this way, there is mutual transformation between material, alcohols, and aldehydes, which are subsequently proceeded to be involved in the formation of esters, or fatty acids proceed through aldehyde dehydrogenase and participate in the fatty acid pathway to form esters. The secondary metabolic pathways of aroma metabolism can be divided into terpene pathways and the synthesis of volatile phenols. In terpene pathways, the main enzyme is terpene synthase, under which semiterpenes, polyterpenes, and other terpenes are synthesized (Portnoy et al., 2008). In addition, shikimic acid can synthesize many aromatic compounds, such as coumarin and flavonoids, through the shikimic acid pathway. Song and Forney et al. found that benzyl acetates are one of the major ester groups in melon, and this ester is usually synthesized through the shikimic acid pathway (Song & Forney, 2008). Additionally, many sulfur compounds contribute to melon flavor, but they have not been identified in "Nasmi" melon in this study due to the fact that the specific melon varieties were used, or the incomplete records in the IMS database.

| Melon aroma content and amount of gene expression in different regions
Aroma content is regulated by a variety of different genes. In this study, transcriptome technology was used to identify the key genes responsible for the difference in aroma metabolism in different production areas. Aroma components and relative contents between production areas were determined by the GC-IMS technology.
Combined analysis showed that aroma content was inextricably related to these key genes. In aroma metabolism, pyruvate dehydrogenase is an important enzyme in the process of pyruvate conversion to acetyl-CoA. The expression levels of several differential genes encoding pyruvate in melon from different production areas changed significantly between the two regions. of these key genes, the aroma composition between the two regions shows large differences.
This study focused on the fatty acid and amino acid pathways of melon aroma metabolism. We discovered that the genes primarily involved in aroma metabolism were acetyl-CoA acyltransferase, 3-hydroxyl-CoA dehydrogenase, acyl-CoA oxidase, long-chain acyl-CoA synthetase, aldehyde dehydrogenase, and alcohol dehydrogenase in transcriptome analysis, which is consistent with previous research (Li et al., 2011;Zhang et al., 2017). At the same time, based on several aroma studies and the current study, we preliminarily obtained the synthesis pathways of aroma components, which provides strong support for melon breeding and other related studies.

| CON CLUS ION
By studying the volatile components of the same melon from different production areas, it was observed that there was no difference in the types of volatile components of the melon from the two regions. However, there was a notable difference in the aroma contents. The main components that differed between the two regions were esters, alcohols, and aldehydes. The total aroma of the TT sample was 1.7 times higher than that of the AT sample. In the two regions, with the same variety of melon, transcriptome sequencing analysis produced 9658 different genes, and through the classification based on GO annotations, the three major categories all demonstrated notable amounts of enrichment. Through the analysis of aroma-related pathways and KEGG analysis, a large number of fruit-related metabolic pathways were obtained.
Investigation of aroma and analysis of multiple genes related to regional differences identified the acetyl-CoA acyltransferase, 3-hydroxyl acyl-coenzyme A, dehydrogenase, acyl-coenzyme A oxidase long-chain acyl-coenzyme A synthetase, acetaldehyde dehydrogenase, and alcohol dehydrogenase genes. Some differentially expressed genes were verified by real-time fluorescence PCR. The results showed that the transcriptome sequencing results were reliable.

ACK N OWLED G M ENTS
This study was funded by the special project for basic scientific activities of non-profit institutes supported the government of Xinjiang Uyghur Autonomous Region (KY2021118 and KY2020108),

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.